The focus of this research proposal is the development of a tissue engineered ligament graft for ACL regeneration. Currently, ACL injuries are the most common knee injury in the United States. As the population places more emphasis on physical activity to combat the obesity epidemic and the level of intensity in individual sports increases, the number of ACL injuries is likely to rise. Current reconstruction strategies are not sufficient for successful long-term clinical outcomes and a new method to repair the ACL-deficient knee is needed. An ACL reconstruction that results in an increased quality of life for the patient may be possible through tissue engineering techniques. Our cell-based approach utilizes mesenchymal stem cells (MSCs), a progenitor cell source suitable for musculoskeletal tissue engineering strategies. By directing lineage commitment of MSCs, a suitable tissue can be formed. We propose to engineer ligament tissue by combining three techniques: electrospun fiber meshes, mechanical stretch and scleraxis-expresssing MSCs. We hypothesize that the fiber architecture of the biomaterial, namely fiber orientation and diameter, will affect MSC response to mechancial stretch and the subsequent development of extracellular matrix. Further, we postulate that MSCs with constitutive overexpression of the ligament/tendon transcription factor, scleraxis, will act synergistically with electrospun biomaterials to promote ligament tissue development. Therefore, we propose two specific aims: (1) To determine the effect of fiber architecture under mechanical stretch on ligament tissue formation. (2) To determine the potential of scleraxis-modified mesenchymal stem cells (MSCs) cultured on electrospun polymers on the formation of ligament tissue. Stem cell differentiation to ligament tissue phenotype will be characterized by gene expression and extracellular matrix protein deposition. The long term goal of this project is an engineered ligament capable of integration and regeneration once implated at the site of injury. The studies outlined in this proposal will generate a platform for developing functional ligaments using a multidimensional approach involving polymeric biomaterial architecture and mechanical stretch as well as genetic engineering.